Our objective is to test the hypothesis that the Ca ions regulatory mechanism for actin-myosin interactions in vascular smooth muscle involves Ca ions dependent phosphorylation of the myosin light chains. The hypothesis suggests that effects of changes in pH on contractility of coronary arterial smooth muscle are partly ascribable to modulation of the phosphorylation-dephosphorylation cycle. We've shown that Ca ions dependent phosphorylation of the light chains occurs in actomyosin from coronary, aortic and carotid smooth muscle and that this is related to actin-mediated activation of Mg ions actomyosin ATPase. By employing a new technique developed in our laboratory which rapidly and quantitatively distinguishes between phosphorylated and nonphosphorylated light chains we will study relationships between contraction of coronary arterial smooth muscle, changes in pH, Mg ions ATPase, and the phosphorylation-dephosphorylation cycle. We also plan on isolating and characterizing the Ca ions dependent kinase responsible for phosphorylation and the phosphatase responsible for dephosphorylation. Emphasis is placed on relating binding of Ca ions to actomyosin, Ca ions, dependent kinase, chemically skinned smooth muscle strips and enzymatic activity (ATPase) or mechanical responses (isometric force) resulting from actin-myosin interactions. Particular attention will be directed to identifying steps involving cooperative interactions between Ca ions and the regulatory proteins. We also have evidence suggesting that a Ca ions sensitive protein resembling TnC or the calcium regulatory protein for phosphodiesterase exists in coronary smooth muscle. We will study how this protein influences the Ca ions regulatory process, particularly as it might relate to light chain phosphorylation. Thus our studies involve experiments with contractile and regulatory proteins isolated from vascular smooth muscle experiments with model systems of smooth muscle that can generate force, and experiments with intact strips of coronary muscle. These studies will provide a better understanding of the molecular mechanisms underlying arterial contractility and how such mechanisms are altered during disease.